Nodulation, which is considered the predominant defense reaction to infection in insects, is a complex process influenced by various endogenous factors. However, the precise mechanisms underlying nodulation remain largely unknown. In the present study, we examined the influence of the insect hormones 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the laminarin-induced nodulation reaction in larvae of the flesh fly Neobellieria bullata. Treating third-instar larvae of N. bullata with 20E prior to laminarin injection enhanced the nodulation response in a dose-dependent manner. The ecdysone agonists RH2485, RH5849 and RH0345 similarly enhanced the nodulation reaction, although they were less active than 20E. In contrast to ecdysone stimulation, supplying larvae with JH or the juvenile hormone analogs (JHA), fenoxycarb and pyriproxyfen, significantly impaired their ability to form nodules in response to laminarin. These findings demonstrate for the first time that 20E and JH play an important regulatory role in the nodulation process.